0.5
A B
0.4 0.3 0.2 0.1 0.0
10 100 1000 1 10 100 1000
Concentration (mg/mL) Concentration (mg/mL)
0.0
Absorbance (A.U.)
Absorbance (A.U.)
0.1 0.2 0.3 0.4 0.5
Fig. 3 Concentration-response curves of HepG2 cells exposed for 24 h to (a) acetaminophen and (b) acetylsali- cylic acid
Neutral Red Uptake Assay
plate by a flip movement into a recipient with large opening or the sink. Eventual liquid at the edges of the plates can be dried by pressing the plate to a pile of paper cloths. This procedure can only be performed if no further culturing of the cells is envisaged.
References
1. Borenfreund E, Puerner J (1984) A simple quantitative procedure using monolayer cul- tures for cytotoxicity assays (HTD/NR90).
J Tissue Cult Methods 9(1):7–9
2. Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3(7):
1125–1131
3. Zuang V (2001) The neutral red release assay:
a review. Altern Lab Anim 29(5):575–599 4. Rodrigues RM, Bouhifd M, Bories G, Sacco
M, Gribaldo L, Fabbri M, Coecke S, Whelan MP (2013) Assessment of an automated in vitro basal cytotoxicity test system based on metabolically-competent cells. Toxicol In Vitro 27(2):760–767
5. The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (2003) Test method protocol for the BALB/c 3T3 neutral red uptake cytotoxicity test. A test for basal cyto- toxicity for an in vitro validation study phase III. https://ntp.niehs.nih.gov/iccvam/meth- ods/acutetox/invidocs/phiiiprot/3t3phiii.
pdf. Accessed Jun 2016
6. Organisation for Economic Co-operation and Development (2004) Guideline 432: in vitro 3T3 NRU phototoxicity test. OECD guidelines for the testing of chemicals. http://www.
oecd-ilibrar y.org/environment/test-no-
432-in-vitro-3t3-nru-phototoxicity- test_
9789264071162-en. Accessed Jun 2016 7. European Commission Joint Research Center
(2013) EURL ECVAM recommendation on the 3T3 neutral red uptake (3T3 NRU) cyto- toxicity assay for the identification of sub- stances not requiring classification for acute oral toxicity. https://eurl-ecvam.jrc.ec.europa.
eu/eurl-ecvam-recommendations/3t3-nru- recommendation. Accessed Jun 2016
8. Bouhifd M, Bories G, Casado J, Coecke S, Norlén H, Parissis N, Rodrigues RM, Whelan MP (2012) Automation of an in vitro cytotox- icity assay used to estimate starting doses in acute oral systemic toxicity tests. Food Chem Toxicol 50(6):2084–2096
9. Knowles BB, Aden DP (1983) Human hepa- toma derived cell line, process for preparation thereof, and uses therefor. US Patent 4,393,133 10. Schoonen WG, Westerink WM, de Roos JA,
Débiton E (2005) Cytotoxic effects of 100 ref- erence compounds on HepG2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I mechanistic assays on ROS, glutathione depletion and calcein uptake. Toxicol In Vitro 19(4):505–516
11. Chiu JH, Hu CP, Lui WY, Lo SJ, Chang CM (1990) The formation of bile canaliculi in human hepatoma-cell lines. Hepatology 11:834–842
27
Chapter 3
Assessment of Cell Viability with Single-, Dual-, and Multi- Staining Methods Using Image Cytometry
Leo Li-Ying Chan, Kelsey J. McCulley, and Sarah L. Kessel
Abstract
The ability to accurately measure cell viability is important for any cell-based assay. Traditionally, viability measurements have been performed using the trypan blue exclusion method on a hemacytometer, which allows researchers to visually distinguish viable from nonviable cells. While the trypan blue method can work for cell lines or primary cells that have been rigorously purified, in more complex samples such as PBMCs, bone marrow, whole blood, or any sample with low viability, this method can lead to errors. In recent years, advances in optics and fluorescent dyes have led to the development of automated benchtop image-based cell counters for rapid cell concentration and viability measurement. In this work, we demon- strate the use of image-based cytometry for cell viability detection using single-, dual-, or multi-stain techniques. Single-staining methods using nucleic acid stains such as EB, PI, 7-AAD, DAPI, SYTOX Green, and SYTOX Red, and enzymatic stains such as CFDA and Calcein AM, were performed. Dual- staining methods using AO/PI, CFDA/PI, Calcein AM/PI, Hoechst/PI, Hoechst/DRAQ7, and DRAQ5/DAPI that enumerate viable and nonviable cells were also performed. Finally, Hoechst/Calcein AM/PI was used for a multi-staining method. Fluorescent viability staining allows exclusion of cellular debris and nonnucleated cells from analysis, which can eliminate the need to perform purification steps during sample preparation and improve efficiency. Image cytometers increase speed and throughput, cap- ture images for visual confirmation of results, and can greatly simplify cell count and viability measurements.
Keywords Image cytometry, Viability, Enzymatic stain, Nucleic acid stain, Multi-stain method, Fluorescent stain, Trypan blue, Cellometer, Celigo
1 Introduction
It is important to accurately measure cell viability for any cell- based assay performed in immuno-oncology, stem cell, and toxi- cology research, or for traditional cell culture and plating for downstream assays [1, 2]. In the past decade, a new generation of affordable chip-based image cytometry systems, such as the Cellometer [3–5] (Nexcelom Bioscience), Countess II [6] (Life Technologies), and NucleoCounter [7] (Chemometec), have been introduced to address the known issues of traditional cell
Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_3, © Springer Science+Business Media LLC 2017
viability detection methods such as manual counting and flow cytometry [8–10]. Manual counting using a hemacytometer is time consuming and has high operator-dependent variations [11]. Flow cytometry systems require a considerable amount of maintenance and highly skilled operators. In addition, the lack of imaging capability may generate uncertainties in the results [12, 13]. In contrast, automated image cytometers can quickly and easily capture and analyze bright-field and fluorescent images of trypan blue (TB) or fluorescently stained target cells to measure the number of live and dead cells and determine viability [14–
17]. High-throughput plate- based image cytometry systems such as the Celigo [18] (Nexcelom Bioscience), Opera [19, 20]
(Perkin Elmer), and IN Cell Analyzer 2200 [20, 21] (GE) have also been developed to measure cell viability in standard multi- well microplates. These high-throughput image cytometers can be used to screen potential cancer drug candidates for drug dis- covery research.
Depending on the cell sample, image cytometers are used to measure cell viability by staining cells with one, two, or three dyes for optimal measurements. For cell lines with high viability or pri- mary cells that have been rigorously purified, a single-dye staining method such as trypan blue or propidium iodide can be used, where the image cytometer measures total and dead cell counts in bright-field and fluorescent images [22–24]. In contrast, primary cell samples that contain a high level of red blood cells (RBC) and platelets require staining with multiple dyes. The total, live, and dead cell counts are measured in fluorescent images, which elimi- nates the potential of counting nonnucleated cells or nonspecific particles in the samples.
In this work, we describe the Cellometer (chip-based) and Celigo (plate-based) image cytometry protocols to rapidly assess cell viability using trypan blue, fluorescent nucleic acid, and enzymatic dyes, as well as utilizing dual- and multi-fluorescent staining methods. Jurkat cells were stained with ethidium bro- mide (EB), propidium iodide (PI), 7-aminoactinomycin D (7-AAD), 4 ′ ,6-diamidino-2-phenylindole (DAPI), SYTOX Green, or SYTOX Red nucleic acid dyes to measure cell viability [4, 25–29]. Similarly, Jurkat cells were stained with carboxyfluo- rescein diacetate (CFDA) or Calcein AM enzymatic dyes to mea- sure cell viability [4, 27, 30]. Next, Jurkat cells were dual-stained with acridine orange, Hoechst 33342 (Hoechst), CFDA, or Calcein AM in combination with PI to enumerate live and dead cells [31–34]. In addition, DRAQ5™ [35] and DRAQ7™ [36]
were used in combination with DAPI and Hoechst, respectively, to stain MCF7 GFP cells. Finally, Hoechst, Calcein AM, and PI were used to stain HeLa cells to measure total, live, and dead cells, respectively [37].
29
2 Materials